PROJECT SUMMARY Progressive and irreversible deterioration of articular cartilage is a central event in osteoarthritis (OA) and other forms of degenerative joint disease (DJD), but despite the high prevalence of these painful and disabling conditions, no treatment is yet available to effectively protect and restore healthy tissue. Cartilage breakdown occurs as a result of both increased tissue catabolism and impaired anabolism. Strategies are being developed to block catabolism, but major knowledge gaps preclude the design of strategies to restore proper cartilage anabolism. This project is designed to fill key gaps. One gap is on the exact roles of the SOX9 transcription factor in adult articular cartilage. SOX9 drives chondrocyte specification and differentiation in developmental chondrogenesis, but its roles in adult cartilage homeostasis and pathology remain unclear. Another gap is on the role of SOX8, the closest relative of SOX9, in cartilage. New data reveal that SOX8 is co-expressed with SOX9 in articular chondrocytes and contributes to adult articular cartilage integrity. Pro-inflammatory and other pathways driving DJD elicit chondrocyte catabolic actions and downregulate cartilage-specific anabolism, including SOX9 expression, and in vitro studies suggest that the catabolic response of chondrocytes to pro- inflammatory cytokines is greatly reduced upon forced expression of SOX8 or SOX9. Together, these data suggest the innovative hypothesis that SOX8 and SOX9 share key roles in articular chondrocytes and that their pathogenic downregulation exacerbates cartilage anabolism repression and catabolism stimulation. Three specific aims are proposed to test this hypothesis. Aim 1 is to define the roles of SOX8 and SOX9 in adult articular chondrocytes in healthy conditions. Either or both genes will be knocked down in human primary articular chondrocytes and conditionally inactivated in articular chondrocytes in adult mice, and consequences will be assessed. Aim 2 is to test whether SOX8 and SOX9 repression impacts OA development. SOX8 and SOX9 expression and activities will be examined for correlations with OA severity in humans. Also, OA will be induced in adult mice and the effect of SOX8/SOX9 inactivation on disease development will be analyzed and mechanistically dissected. Aim 3 is to test whether and how forced expression of SOX8 or SOX9 helps maintain articular chondrocyte normalcy in OA conditions. The SOX proteins will be overexpressed in primary human articular chondrocytes from normal and OA knees and their impact on cell behavior assessed. Mice will be conditionally forced to express the SOX proteins in articular chondrocytes and will be tested for cartilage fate under normal and OA conditions. A team of expert investigators and clinicians has been assembled to ensure the successful achievement of the project. Novel discoveries are expected to transform current understanding of key mechanisms dictating articular chondrocyte specification and activities in health and disease, and thereby to spark novel ideas for the design of highly needed effective treatments for the millions of patients suffering from OA and other joint degenerative diseases around the world.